Citation: Cell Death and Disease (2017) 8, e2866; doi:10.1038/cddis.2017.257 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis ARRDC4 regulates enterovirus 71-induced innate immune response by promoting K63 polyubiquitination of MDA5 through TRIM65 Jun Meng1,2,6, Zhenyu Yao3,6, Yaqing He1, Renli Zhang1, Yanwei Zhang1, Xiangjie Yao1, Hong Yang1, Long Chen1, Zhen Zhang1, Hailong Zhang1, Xueqin Bao4, Gang Hu5, Tangchun Wu*,2 and Jinquan Cheng*,1 Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease (HFMD), which induces significantly elevated levels of cytokines and chemokines, leading to local or system inflammation and severe complications, whereas the underlying regulatory mechanisms and the inflammatory pathogenesis remain elusive. ARRDC4 is one member of arrestins family, having important roles in glucose metabolism and G-protein-coupled receptors (GPCRs) related physiological and pathological processes, however, the function of ARRDC4 in innate immune system is largely unknown. Here we identified that ARRDC4 expression was increased after EV71 infection in THP-1-derived macrophages and verified in EV71-infected HFMD patients and the healthy candidates. The expression level of ARRDC4 was positively correlated with the serum concentration of IL-6, TNF-α and CCL3 in clinical specimens. ARRDC4 interacted with MDA5 via the arrestin-like N domain, and further recruited TRIM65 to enhance the K63 ubiquitination of MDA5, resulting in activation of the downstream innate signaling pathway and transcription of proinflammatory cytokines during EV71 infection. Our data highlight new function of ARRDC4 in innate immunity, contributing to the better understanding about regulation of MDA5 activation after EV71 infection, and also suggest ARRDC4 may serve as a potential target for intervention of EV71-induced inflammatory response. Cell Death and Disease (2017) 8, e2866; doi:10.1038/cddis.2017.257; published online 8 June 2017 Enterovirus 71 (EV71) is a single positive-stranded RNA virus detect long fragments viral RNA (44kb) mainly from picorna- belonging to the picornaviridae family. EV71 is the main virus and paramyxovirus.8–10 Potentiated by E3 ligase-mediated causative agent of hand, foot and mouth disease (HFMD), K63 polyubiquitination, RIG-I and MDA5 are activated and which induces a wide range of clinical manifestations leading to subsequently activate the essential adaptor protein mitochon- severe neurological complications in infants and children drial antiviral signaling (MAVS). MAVS further recruits down- younger than 5 years old.1 EV71-infected patients carry high stream adaptors to activated transcription factors IFN regulatory level of cytokines and chemokines,2,3 whereas exhibit impaired factors (IRFs), NF-κB and activator protein 1 (AP-1), inducing production of type I interferon (IFN).4,5 In the absence of type I production of proinflammatory cytokines and type I IFNs.11 IFNs, the other cytokines and chemokines are irreplaceable to RLR-triggered innate immune signaling is tightly regulated defense against EV71 invasion, however, the disordered to achieve effective viral clearance without immunopathology. production of inflammation mediators may lead to cell or tissue Polyubiquitination is one of the key regulatory mechanisms for injury. The detailed regulation mechanism and the inflammatory RLRs. Upon recognition of viral RNA, RIG-I undergoes K63 pathogenesis after EV71 infection remain elusive. polyubiquitination catalyzed by tripartite motif protein 25 or Once viruses invade the host, the innate immune system Riplet to activate innate signaling pathway,12,13 whereas ring- detects viral nucleic acid with pattern recognition receptors finger protein 125 (RNF125) acts as negative regulators and (PRRs), including Toll-like receptors (TLRs), retinoic acid- mediates K48-linked polyubiquitination of RIG-I and MDA5, inducible gene-I (RIG-I)-like receptors (RLRs) and other leading to their degradation through proteasome.14 The cytosolic sensors.6 The crucial members of RLRs, RIG-I and regulation of RIG-I ubiquitination has been extensively – melanoma differentiation-associated gene 5 (MDA5), have non- investigated,12 18 however, the regulation mechanism of redundant roles in cytosolic viral RNA sensing.7 MDA5 prefers to MDA5 ubiquitination is poorly understood. 1Department of Microbiology, Shenzhen Center for Disease Control and Prevention, Shenzhen, Guangdong, China; 2Department of Occupational and Environmental Health Key Laboratory of Environment and Health, Ministry of Education and State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; 3Department of Translational Medicine R&D Center, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, China; 4Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China and 5Department of Biostatistics and Epidemiology, College of Public Health, Sun Yat-SenUniversity, Guangzhou, Guangdong, China *Corresponding author: T Wu, Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. Tel: +86 27 8369 2333; Fax: +86 27 8369 2560; E-mail: [email protected] or J Cheng, Shenzhen Center for Disease Control and Prevention, Shenzhen, Guangdong, China. Tel: +86 755 2562 6488; Fax: +86 755 2553 2595; E-mail: [email protected] 6These authors contributed equally to this work. Received 17.1.17; revised 16.4.17; accepted 04.5.17; Edited by A Oberst ARRDC4 promotes EV71-induced MDA5 activation JMenget al 2 Figure 1 EV71 infection upregulates ARRDC4 expression. (a) Microarray analysis of EV71-infected t-MØ (MOI = 50) for indicated time. Heat map represents arrestins family genes expression change (Po0.05). (b) Q-PCR analysis of expression of α-arrestin members in t-MØ treated with EV71 for 24 h or untreated. (c) mRNA expression of ARRDC4 was assayed by Q-PCR in t-MØ infected with EV71 for indicated time. (d) Immunoblot analysis of ARRDC4, EV71-VP1 and β-actin expression in lysates of t-MØ stimulated with EV71 for shown time. Data are shown as mean ± S.D. of triplicate samples (b and c), or are representative of three independent experiments with similar results (d) Arrestins family consists of visual arrestins (S antigen (SAG) Results and arrestin 3 (ARR3)), β-arrestin 1–2 (ARRB1 and ARRB2), and α-arrestins (arrestin domain-containing 1–5 (ARRDC1–5) ARRDC4 expression is upregulated in response to EV71 and thioredoxin-interacting protein (TXNIP)), which have infection. In order to identify molecules selectively involved essential and versatile roles in the signaling of G-protein- in the regulation of EV71-triggered innate immune response, coupled receptors (GPCRs).19 In recent years, some mem- we screened differential expression molecules in EV71- bers of the arrestins family have been reported involving in infected THP-1-derived macrophages (t-MØ) through Gene- more diversiform physical and pathological processes. Much Chip Microarrays, and identified all the members of arrestins of the concerns are about the β-arrestins, especially in family were up or downregulated (Figure 1a). ARRDC4 was immune and inflammatory responses.20 β-Arrestin can nega- the most significantly upregulated after EV71 infection tively regulate LPS triggered proinflammatory cytokines (Figure 1a). We validated the expression of α-arrestins 21 production, and have moderates roles in antiviral innate subfamily members in EV71-infected t-MØ by Q-PCR, and immune response.22 ARRDC4 is one member of α-arrestins, got the same results as the microarrays (Figure 1b). More- which has the arrestin-like structure and highly conserved over, the mRNA and protein levels of ARRDC4 were polyproline (PY) motif in the C-terminal tail.19 ARRDC4 increased in response to EV71 infection in a time- participates in glucose metabolism,23 and exhibits more varied dependent manner (Figures 1c and d). These results indicate functions through recruiting some E3 ligases to induce that ARRDC4 may be involved in EV71-induced immune ubiquitination of GPCRs.24 Interestingly, it has been sug- response. gested that α-arrestin and β-arrestin may hetero-associate and have coordinated or antagonistic functions depending on ARRDC4 promotes EV71-triggered proinflammatory context,25 however, the function of ARRDC4 in immune cytokines production. To identify the role of ARRDC4 in response is largely unknown, thus deserves fuller exploration. EV71-triggered immune responses, we silenced ARRDC4 Herein, we analyzed gene expression profiles in EV71- with specific siRNA in t-MØ, and detected significantly infected macrophages with GeneChip Microarrays, notably, all knockdown of ARRDC4 expression in both mRNA and of the arrestins family members were up or down regulated, protein levels (Figures 2a and b). ARRDC4 silencing and ARRDC4 exhibited the highest elevated level. In EV71- antagonized the mRNA expression and the production of infected clinical specimens, the expression level of ARDDC4 IL-6, TNF-α and CCL3 in t-MØ infected with EV71 (Figures 2c was positively correlated with the serum concentration of and d). However, type I IFN (IFN-α and IFN-β) expression in proinflammatory